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Cryo-EM structure of the bacteriophage T4 portal protein assembly at near-atomic resolution.

Sun L, Zhang X, Gao S, Rao PA, Padilla-Sanchez V, Chen Z, Sun S, Xiang Y, Subramaniam S, Rao VB, Rossmann MG - Nat Commun (2015)

Bottom Line: However, the detailed structure of the portal protein remained unknown.The gp20 structure also verifies that the portal assembly is required for initiating head assembly, for attachment of the packaging motor, and for participation in DNA packaging.Comparison of the Myoviridae T4 portal structure with the known portal structures of φ29, SPP1 and P22, representing Podo- and Siphoviridae, shows that the portal structure probably dates back to a time when self-replicating microorganisms were being established on Earth.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 240S. Martin Jischke Drive, West Lafayette, Indiana 47907-2032, USA.

ABSTRACT
The structure and assembly of bacteriophage T4 has been extensively studied. However, the detailed structure of the portal protein remained unknown. Here we report the structure of the bacteriophage T4 portal assembly, gene product 20 (gp20), determined by cryo-electron microscopy (cryo-EM) to 3.6 Å resolution. In addition, analysis of a 10 Å resolution cryo-EM map of an empty prolate T4 head shows how the dodecameric portal assembly interacts with the capsid protein gp23 at the special pentameric vertex. The gp20 structure also verifies that the portal assembly is required for initiating head assembly, for attachment of the packaging motor, and for participation in DNA packaging. Comparison of the Myoviridae T4 portal structure with the known portal structures of φ29, SPP1 and P22, representing Podo- and Siphoviridae, shows that the portal structure probably dates back to a time when self-replicating microorganisms were being established on Earth.

No MeSH data available.


Cryo-EM structure of the T4 portal protein (gp20) assembly.(a) 3D density map of T4 portal protein assembly at 3.6 Å resolution with each subunit colour-coded. Shown are the top view (left) and side view (right). (b) Ribbon diagram of the gp20 atomic model with each subunit colour-coded. Shown are the top view (left) and side view (right).
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f1: Cryo-EM structure of the T4 portal protein (gp20) assembly.(a) 3D density map of T4 portal protein assembly at 3.6 Å resolution with each subunit colour-coded. Shown are the top view (left) and side view (right). (b) Ribbon diagram of the gp20 atomic model with each subunit colour-coded. Shown are the top view (left) and side view (right).

Mentions: The gp20-N74 protein was used to collect EM data with an FEI Titan Krios microscope equipped with a Gatan K2 Summit direct electron detector. A total of about 98,000 particles were boxed from 839 selected averaged images (see Methods). Aberrant particles could be recognized and then discarded in the two-dimensional classification procedure. The reconstruction assumed 12-fold symmetry. An early difficulty in determining the structure was the preference by the particles to orient themselves with their 12-fold axes perpendicular to the ice surface, resulting in a dearth of side views. This was partially rectified by adding octyl-β-glucoside to the sample before freezing (Supplementary Fig. 1a,b). The final three-dimensional cryo-EM reconstruction had an overall resolution of 3.6 Å (Fig. 1a), based on the ‘gold-standard' Fourier shell correlation (FSC) using the FSC=0.143 criterion (Supplementary Fig. 1e). The map showed continuous density for the polypeptide chains and recognizable side-chain densities (Supplementary Fig. 2). An atomic model was built based on this map, using the COOT graphical program30. The crystallographic program Phenix31 were used to refine the model (Fig. 1b) by minimizing the vector difference between the Fourier coefficients obtained by back-transforming the final cryo-EM map and the calculated structure factors derived from the atomic model. The final R (working) and R (free) values were 0.26 and 0.27, respectively (Supplementary Table 2).


Cryo-EM structure of the bacteriophage T4 portal protein assembly at near-atomic resolution.

Sun L, Zhang X, Gao S, Rao PA, Padilla-Sanchez V, Chen Z, Sun S, Xiang Y, Subramaniam S, Rao VB, Rossmann MG - Nat Commun (2015)

Cryo-EM structure of the T4 portal protein (gp20) assembly.(a) 3D density map of T4 portal protein assembly at 3.6 Å resolution with each subunit colour-coded. Shown are the top view (left) and side view (right). (b) Ribbon diagram of the gp20 atomic model with each subunit colour-coded. Shown are the top view (left) and side view (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493910&req=5

f1: Cryo-EM structure of the T4 portal protein (gp20) assembly.(a) 3D density map of T4 portal protein assembly at 3.6 Å resolution with each subunit colour-coded. Shown are the top view (left) and side view (right). (b) Ribbon diagram of the gp20 atomic model with each subunit colour-coded. Shown are the top view (left) and side view (right).
Mentions: The gp20-N74 protein was used to collect EM data with an FEI Titan Krios microscope equipped with a Gatan K2 Summit direct electron detector. A total of about 98,000 particles were boxed from 839 selected averaged images (see Methods). Aberrant particles could be recognized and then discarded in the two-dimensional classification procedure. The reconstruction assumed 12-fold symmetry. An early difficulty in determining the structure was the preference by the particles to orient themselves with their 12-fold axes perpendicular to the ice surface, resulting in a dearth of side views. This was partially rectified by adding octyl-β-glucoside to the sample before freezing (Supplementary Fig. 1a,b). The final three-dimensional cryo-EM reconstruction had an overall resolution of 3.6 Å (Fig. 1a), based on the ‘gold-standard' Fourier shell correlation (FSC) using the FSC=0.143 criterion (Supplementary Fig. 1e). The map showed continuous density for the polypeptide chains and recognizable side-chain densities (Supplementary Fig. 2). An atomic model was built based on this map, using the COOT graphical program30. The crystallographic program Phenix31 were used to refine the model (Fig. 1b) by minimizing the vector difference between the Fourier coefficients obtained by back-transforming the final cryo-EM map and the calculated structure factors derived from the atomic model. The final R (working) and R (free) values were 0.26 and 0.27, respectively (Supplementary Table 2).

Bottom Line: However, the detailed structure of the portal protein remained unknown.The gp20 structure also verifies that the portal assembly is required for initiating head assembly, for attachment of the packaging motor, and for participation in DNA packaging.Comparison of the Myoviridae T4 portal structure with the known portal structures of φ29, SPP1 and P22, representing Podo- and Siphoviridae, shows that the portal structure probably dates back to a time when self-replicating microorganisms were being established on Earth.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 240S. Martin Jischke Drive, West Lafayette, Indiana 47907-2032, USA.

ABSTRACT
The structure and assembly of bacteriophage T4 has been extensively studied. However, the detailed structure of the portal protein remained unknown. Here we report the structure of the bacteriophage T4 portal assembly, gene product 20 (gp20), determined by cryo-electron microscopy (cryo-EM) to 3.6 Å resolution. In addition, analysis of a 10 Å resolution cryo-EM map of an empty prolate T4 head shows how the dodecameric portal assembly interacts with the capsid protein gp23 at the special pentameric vertex. The gp20 structure also verifies that the portal assembly is required for initiating head assembly, for attachment of the packaging motor, and for participation in DNA packaging. Comparison of the Myoviridae T4 portal structure with the known portal structures of φ29, SPP1 and P22, representing Podo- and Siphoviridae, shows that the portal structure probably dates back to a time when self-replicating microorganisms were being established on Earth.

No MeSH data available.